NO acts as a chemical messenger in the control of many important processes in vivo, including vasodilation, neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. Since the discovery in 1867 by Drs. Lauder Brunton and William Murrell that nitroglycerin (GTN) is capable of treating heart disease conditions such as angina pectoris, organic nitrates have been widely used to treat acute cases of vasoconstriction. Within the last several decades, the mechanism of vasodilation has been elucidated. NO, which is synthesized in endothelial cells, diffuses to smooth muscle cells and activates soluble guanylate cyclase (sGC) to produce cyclic GMP, and thereby induce vasodilation. The clinical mechanism of action of organic nitrates, then, is presumed to require their biotransformation to NO and subsequent activation of sGC. However, organic nitrates cease to be effective in patients after 24-48 hours, due to a phenomenon called tolerance. Thus, for treatment of chronic cases of hypertension, compounds such as β-blockers and ACE inhibitors are used, although they too have limitations and side effects. Thus, nitrovasodilators are most useful in treating acute situations where rapid vasodilation is required to alleviate symptoms such as angina and myocardial infarction. Prolonged administration of organic nitrates results in reduced efficacy, and the vasculature becomes non-responsive; this tolerance prevents their further use both in chronic and acute cases. Thus, for acute treatment, non-continuous nitrovasodilator use is employed with limited effect. For chronic cases of vasoconstriction, other avenues of treatment are employed, typically using a mixed regimen of organic nitrates and NO-independent blood pressure medications, with mixed success.
Two major competing theories on the mechanism for tolerance run parallel to the search for the mechanism of biotransformation of nitrates that leads to release of NO. Because NO is believed to be the mediator of the vasodilatory effects of organic nitrates, the mechanism of release of NO from organic nitrates may become inhibited, resulting in tolerance. But how organic nitrates metabolically release NO in tissues is not understood. Furthermore, the mechanism-based theory for tolerance is problematic because tolerance also reduces the efficacy of endogenous NO and exogenous NO gas in mediating vasodilation. Thus, the mechanism of biotransformation of organic nitrates appears to be separate from the reason for tolerance. A competing theory posits that the response to NO from organic nitrates becomes dampened in the target tissue, perhaps because the generation of NO and the by-products of the reaction eventually inhibit the response to NO, or because acute activation of the NO pathway has a feedback mechanism that desensitizes it to further stimulation. This theory is known as end-organ tolerance. Recently, a unifying theory has been proposed that includes aspects of the biotransformation of organic nitrates as well as end-organ desensitization to NO. Essentially, biotransformation of organic nitrates appears to result in higher levels of superoxide (O2−) in tissues. Superoxide reacts at the rate of diffusion with NO to produce peroxynitrite (OONO). This reaction essentially traps and destroys basal NO, preventing it from activating sGC. Reduced NO levels leads to vasoconstriction, and OONO− is a powerful oxidant that damages tissues. Prolonged treatment with organic nitrates such as GTN can result in hypertension and tissue damage in patients, and this can be moderated with co-administration of antioxidants such as ascorbate. Thus, improved therapeutics for delivering NO to organs and tissues to alleviate vasoconstriction is a major therapeutic goal.
Some research has been conducted on the use of hemoglobin-based carriers to deliver NO. However, hemoglobin-based carriers are limited due to their reactivity with NO in the presence of O2, which leads to the inactivation of hemoglobin-based carriers. NO reacts directly with O2 that is bound to hemoglobin to form methemoglobin and nitrate. Both the heme iron and NO become oxidized by the bound oxygen atoms, and the reaction occurs so rapidly that no replacement of O2 by NO is observed (see, e.g., U.S. Pat. No. 6,455,676).
Since NO is produced and consumed on a continuous basis, there is a natural turnover of NO in vivo. When cell-free hemoglobin is administered, the balance between NO production and consumption is altered by reactions with cell-free hemoglobin. The oxidative reaction between NO and O2 bound to hemoglobin is irreversible, resulting in the destruction of NO, O2, and hemoglobin. NO binding to hemoglobin without O2 bound is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours, thereby effectively inactivating hemoglobin as a cell-free NO carrier. Once an NO molecule reacts, with hemoglobin, it is eliminated from the pool of signal molecules, thereby causing certain adverse conditions. For example, the binding of NO to hemoglobin (with or without O2 bound) can prevent vascular relaxation and potentially lead to hypertension, which is sometimes observed after the administration of certain extracellular hemoglobin solutions.
NO is also needed to mediate certain inflammatory responses. For example, NO produced by the endothelium inhibits platelet aggregation. Consequently, as NO is bound by cell-free hemoglobin (with or without O2 bound), platelet aggregation may increase. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A2 and serotonin. These compounds may act synergistically with the reduced NO levels caused by hemoglobin scavenging to produce significant vasoconstriction. In addition to inhibiting platelet aggregation, NO also inhibits neutrophil attachment to cell walls, which in turn can lead to cell wall damage. Endothelial cell wall damage has been observed with the infusion of certain hemoglobin solutions. Hemoglobin-based NO carriers are also hindered by the rapid clearance of cell-free hemoglobin from plasma due the presence of receptors for hemoglobin that remove cell-free hemoglobin from plasma. Cell-free hemoglobin may also cause kidney toxicity, possibly due to NO depletion in glomeruli, causing constriction and subsequent dysfunction.
Due to the limitations of current nitrovasodilator therapies, there remains a significant interest in and need for additional or alternative therapies for delivering NO. In particular, NO carriers that produce less tolerance are needed. Additionally, NO carriers with a low rate of inactivation by NO in the presence of O2 are desired, such as NO carriers that have a low NO reactivity and/or a low affinity for O2. NO carriers with NO dissociation constants or NO dissociation rates that are appropriate for particular clinical or industrial applications are also needed.